The present invention, in some embodiments thereof, relates to an optical resonance analysis system for analyzing reactions of chemical, biochemical, and/or biological materials and detecting properties of the above reactions. The present invention may be employed by types of optical resonance analysis systems in which light wave properties are measured as a function of angle of incidence, reflection, and or refraction. These optical systems include ellipsometry sensors, total internal reflectance (TIR) sensors, and SPR sensors, Brewster angle sensors.
Ellipsometry relies on the change of polarization of light waves reflected off a surface which is in contact with a specimen, to yield information about the specimen itself. Ellipsometry can probe the complex refractive index or dielectric function tensor, which gives access to fundamental physical parameters and is related to a variety of specimen properties, including morphology, crystal quality, chemical composition, or electrical conductivity.
In a standard ellipsometry analysis system, a light wave is emitted by a light source and is linearly polarized by a polarizer. The light wave illuminates a reflecting surface, which is in contact with the specimen, at an angle of incidence. The reflected light wave passes an optional compensator and a second polarizer, which is called analyzer, and falls into a detecting unit. The emitted and the reflected waves span the plane of incidence. Light waves, which are polarized parallel or perpendicular to the plane of incidence, are called p or s polarized, respectively. The detecting unit measures the ratio of the s-polarized and p-polarized components of the reflected light wave. Changes of this ratio give an insight upon the properties of the specimen.
Surface plasmons, are surface electromagnetic waves that propagate parallel along a metal-dielectric or metal-vacuum interface. Since the wave is on the boundary of the metal and the external medium, these oscillations are very sensitive to any change of this boundary, such as the absorption of molecules to the metal surface.
A typical SPR analysis system 100 is described in FIG. 1. A light wave 102 is emitted by a light wave source 104, polarized by a polarizer 106, refracted by a first surface 108 of a prism 110, before reaching a conductive layer 112—usually a layer of gold, silver, or aluminum, characterized by a thickness of about 50 nm—at an angle of incidence φi. At conductive layer 112, some of the energy of light wave 102 is coupled to surface plasmons of conductive layer 112. Therefore, light wave 114, reflected at an angle of reflection φr, by conductive layer 112, has a lower intensity than light waves 102. A detecting unit 116 receives reflected light wave 114, after light wave 114 exits a third surface 118 of prism 110, and measures the intensity of light wave 114. Conductive layer 112 is in contact with a specimen 120, usually a liquid or a gas, held within a specimen channel 121. Optionally, ligands 122 are placed on surface 124 of conductive layer 112. Ligands 122 hold target molecules 126 (also referred to as “analytes”) to test the reaction of specimen 120 with target molecules 126. Changes within specimen 120—such as chemical, biochemical, and biological reactions—affect the behavior of plasmons, and the intensity measurement by detecting unit 116 is used to identify properties of specimen 120.
The energy lost by emitted wave 102 to the coupling with the surface plasmons depends on angle of reflection φr. Generally, for waves of a given wavelength, the energy lost reaches a maximum at an angle, which is called “resonance angle”. The amplitude of the resonance angle changes according to the properties of the specimen. Therefore, for a fixed wavelength, a comparison between the “intensity vs. angle of reflection” graphs of a specimen before and after a reaction, and observation of the shift of the graph over time of the specimen provide data on the reaction—such as association and disassociation rates. FIG. 2 shows “intensity (I) vs. angle of reflection (φr)” graph for the specimen before the reaction (202), and the graph for the specimen after the reaction (204) The shift (206) between curve 202 and curve 204 is also shown. It is noted that φ1 is the resonance angle for the specimen before the reaction, while φ2 is the resonance angle for the specimen after the reaction of the specimen.
Total internal reflection (TIR) is an optical phenomenon that occurs when a light wave strikes a medium boundary at an angle larger than the critical angle with respect to the normal to the surface. If the refractive index is lower on the other side of the boundary no light wave can pass through, so effectively the whole wave is reflected, and an evanescent electromagnetic field is created at the boundary surface. The critical angle is the angle of incidence above which the total internal reflection occurs.
In a typical TIR analysis system, a light source emits a light wave that enters a prism through a first surface, and hits a second surface of the prism at an angle greater than the critical angle. At the second surface, therefore, total internal reflection is achieved, and an evanescent electromagnetic field is created. The light wave is reflected, but its polarization is changed. The second surface of the prism is in contact with a specimen to be assayed, and the properties of the specimen affect the polarization of the reflected light wave. The reflected wave exits the prism through a third surface, and reaches a detecting unit. The detecting unit receives the reflected light wave and measures the polarization of the reflected wave. Properties of the specimen are then calculated by relating the polarization values of the reflected wave to the angle of reflection. Optionally, the detecting unit measures the intensity of the reflected wave, and the properties of the specimen are calculated by an analysis of the total internal reflection step which characterizes a TIR “intensity vs. angle of reflectance” graph.
The Brewster angle is an angle of incidence at which p-polarized light, which illuminates a boundary between two media having different refractive indexes, is not reflected by the boundary. In a typical Brewster angle analysis system, a light source emits a light, which is polarized into p-state by a polarizer. The light illuminates a boundary between two media at angles close to the Brewster angle at which a specimen is introduced, and some of light is reflected toward a detector. Since the Brewster angle is sensitive to changes within the specimen, the polarization of the reflected light is affected by the changes of specimen. The detector measures the polarization of the reflected wave, in order to detect properties of the specimen.
The above systems are largely used in chemistry, biology, and biochemistry, as they are very sensitive to changes in specimens, which makes them suitable for measuring properties of reactions. In the above analysis systems, the reflective surfaces in contact with the specimen may be divided into a plurality of sensing areas, each sensing area in contact with a different specimen. This may allow the analysis systems to perform measurements on a plurality of specimens in a short time. For such a system to be effective, the detector is typically configured to take measurements of light waves characterized by different angles of reflection.
In U.S. Pat. No. 5,327,225 by Bender et al., disclosing a surface plasmon resonance sensor, reflected light from the conductive layer is guided to the detector by a wave guide. In U.S. Pat. No. 6,111,652 by Melendez et al., disclosing a high throughput apparatus for determining interaction properties of test entities, the detector has a large surface for receiving light waves at different angles of reflection. In U.S. Pat. No. 7,218,401 by Iwata et al., disclosing a surface plasmon sensor, a plurality of light detection means is provided, and each one of the detection means is positioned to receive light waves at a predetermined reflective angle. In U.S. Pat. No. 5,035,863 by Finlan et al., disclosing a sequencing apparatus, means for moving the detector are provided in order to enable the detector to take measurements of light waves characterized by different angles of reflection.
U.S. Pat. No. 7,057,720 by Caracci et al., which discloses an optical interrogation system and method capable of generating light beams that have desired optical properties which are directed towards a specimen array, a mask is provided. The mask is located between the detector and the specimen unit, and blocks predetermined light beams reflected from selected specimens in the specimen unit. However, this mask prevents the system described in the invention from assaying different specimens simultaneously.
U.S. patent application Ser. No. 11/274,923 by Takayama et al., discloses an analysis apparatus for analyzing samples by means of detecting lights from a plurality of spots formed on an analysis chip so as to hold the samples. The system includes a selectively light-transmitting unit for transmitting lights selectively from desired spots on a specimen unit to a light-sensitive detector. However, this selectively light-transmitting unit does not allow simultaneous analysis of a plurality of light waves coming from a plurality of areas in the specimen unit.
U.S. Pat. No. 6,873,417 by Bahatt et al. discloses an optical resonance analysis system, which optionally includes an optical system to focus different wavelengths to different areas of the detector.